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Quantitative BONCAT Allows Identification of Newly Synthesized Proteins after Optic Nerve Injury
Sahil H Shah1,2,3, Lucio M Schiapparelli2,4, Satoshi Yokota1
1Mary M. and Sash A. Spencer Center for Vision Research, Byers Eye Institute, Stanford University, Palo Alto, California 94303.
Abstract:
Retinal ganglion cells (RGCs) die after optic nerve trauma or in degenerative disease. However, acute changes in protein expression that may regulate RGC response to injury are not fully understood, and detailed methods to quantify new protein synthesis have not been tested. Here, we develop and apply a new in vivo quantitative measure of newly synthesized proteins to examine changes occurring in the retina after optic nerve injury. Azidohomoalanine, a noncanonical amino acid, was injected intravitreally into the eyes of rodents of either sex with or without optic nerve injury. Isotope variants of biotin-alkyne were used for quantitative BONCAT (QBONCAT) mass spectrometry, allowing identification of protein synthesis and transport rate changes in more than 1000 proteins at 1 or 5 d after optic nerve injury. In vitro screening showed several newly synthesized proteins regulate axon outgrowth in primary neurons in vitro This novel approach to targeted quantification of newly synthesized proteins after injury uncovers a dynamic translational response within broader proteostasis regulation and enhances our understanding of the cellular response to injury.SIGNIFICANCE STATEMENT Optic nerve injury results in death and degeneration of retinal ganglion cells and their axons. The specific cellular response to injury, including changes in new protein synthesis, is obscured by existing proteins and protein degradation. In this study, we introduce QBONCAT to isolate and quantify acute protein synthesis and subsequent transport between cellular compartments. We identify novel candidate protein effectors of the regenerative response and uncover their regulation of axon growth in vitro, validating the utility of QBONCAT for the discovery of novel regulatory and therapeutic candidates after optic nerve injury.
Insights
We developed a new method to measure protein synthesis after optic nerve injury. This technique identified new proteins that regulate retinal ganglion cell axon growth, offering therapeutic targets.
Area of Science:
- Neuroscience
- Cell Biology
- Proteomics
Background:
- Retinal ganglion cells (RGCs) are crucial for vision and vulnerable to optic nerve injury and disease.
- Understanding acute protein synthesis changes post-injury is vital for RGC survival but remains challenging.
- Existing methods struggle to differentiate newly synthesized proteins from existing ones.
Purpose of the Study:
- To develop and validate a novel in vivo method for quantifying acute protein synthesis after optic nerve injury.
- To identify specific proteins synthesized and transported in the retina following optic nerve trauma.
- To explore the role of newly synthesized proteins in RGC axon regeneration.
Main Methods:
- Utilized azidohomoalanine, a noncanonical amino acid, injected intravitreally in rodents with optic nerve injury.
- Employed quantitative bio-orthogonal non-canonical amino acid tagging (QBONCAT) mass spectrometry.
- Analyzed protein synthesis and transport rates for over 1000 proteins at 1 and 5 days post-injury.
Main Results:
- Successfully quantified acute changes in newly synthesized proteins in the retina after optic nerve injury.
- Identified over 1000 proteins with altered synthesis and transport rates.
- Discovered several newly synthesized proteins that promote axon outgrowth in primary neuronal cultures.
Conclusions:
- The QBONCAT method provides a powerful tool to study dynamic translational responses and proteostasis after injury.
- Newly synthesized proteins play a significant role in the cellular response to optic nerve injury.
- Identified novel protein candidates for therapeutic intervention to promote RGC survival and axon regeneration.
